Absence of an Intron Splicing Silencer in Porcine Smn1 Intron 7 Confers Immunity to the Exon Skipping Mutation in Human SMN2
et al. (2014) Absence of an Intron Splicing Silencer in Porcine Smn1 Intron 7 Confers
Immunity to the Exon Skipping Mutation in Human SMN2. PLoS ONE 9(6): e98841. doi:10.1371/journal.pone.0098841
Absence of an Intron Splicing Silencer in Porcine Smn1 Intron 7 Confers Immunity to the Exon Skipping Mutation in Human SMN2
Thomas Koed Doktor 0
Lisbeth Dahl Schrder 0
Henriette Skovgaard Andersen 0
Sabrina Brner 0
Anna Kitewska 0
Charlotte Brandt Srensen 0
Brage Storstein Andresen 0
Emanuele Buratti, International Centre for Genetic Engineering and Biotechnology, Italy
0 1 Department of Biochemistry and Molecular Biology, University of Southern Denmark , Odense M, Denmark , 2 Department of Biomedicine, Aarhus University , Aarhus C, Denmark , 3 Institute of Animal Reproduction and Food Research, Polish Academy of Sciences , Olsztyn , Poland
Spinal Muscular Atrophy is caused by homozygous loss of SMN1. All patients retain at least one copy of SMN2 which produces an identical protein but at lower levels due to a silent mutation in exon 7 which results in predominant exclusion of the exon. Therapies targeting the splicing of SMN2 exon 7 have been in development for several years, and their efficacy has been measured using either in vitro cellular assays or in vivo small animal models such as mice. In this study we evaluated the potential for constructing a mini-pig animal model by introducing minimal changes in the endogenous porcine Smn1 gene to maintain the native genomic structure and regulation. We found that while a Smn2-like mutation can be introduced in the porcine Smn1 gene and can diminish the function of the ESE, it would not recapitulate the splicing pattern seen in human SMN2 due to absence of a functional ISS immediately downstream of exon 7. We investigated the ISS region and show here that the porcine ISS is inactive due to disruption of a proximal hnRNP A1 binding site, while a distal hnRNP A1 binding site remains functional but is unable to maintain the functionality of the ISS as a whole.
Funding: This work was supported by a grant from The Riisfort Foundation (BSA), the Lundbeck foundation (BSA) and The Danish Medical Research Council (FSS
grants no. 271-07-342 and no. 11-107174) to BSA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
The Spinal Muscular Atrophies (SMA) is a phenotypically
diverse but genetically very similar group, in that the diseases are
all caused by homozygous loss of the SMN1 gene . The disease
modifier gene, SMN2, determines to some extent the phenotype of
the affected individual and is unique to the hominid line . As
such, SMA caused by reduced amounts of SMN protein is a
disease unique to humans and the study of animal models is
therefore restricted to transgenic animals. Of these, mouse models
have been used extensively in the past , but the metabolic
and physiological differences between humans and mice are
limiting the potential of the mouse model for evaluation of drug
candidates and studying the molecular pathology of the disease in
detail. Several metabolic and physiological symptoms have been
described in mouse models, which are either rare or only observed
in very severe human cases  or more likely explained by the
genetic background of the particular model . The pig is in
many ways a better model of human biology and mini-pigs are
especially good models since they grow to app. human size and
weight as adults. Pigs are known to be more genetically similar to
humans than mice are , and their metabolism as well as
physiology more close to ours than the mices. For these reasons
we set out to evaluate the potential of constructing a mini-pig
animal model of SMA in order to facilitate improved drug
candidate testing and studies of disease pathology. Furthermore, a
mini-pig model would be extremely valuable in determining the
potential of stem cell treatments as the central nervous system
(CNS) of pigs is very similar to the human CNS. An SMA pig
model would therefore be relevant as an animal model for not only
SMA but also other motor neuron diseases or in the case of
traumatic injury to the motor neurons in the spinal cord.
The role of SMN2 in humans is unclear in the population as a
whole, but in SMA patients SMN2 serves an important function as
the remaining SMN expressing gene. It fails to completely
compensate for the loss of SMN1 due to aberrant splicing of exon
7 which leads to the production of predominantly truncated
transcripts and a corresponding decrease in the amounts of
functional protein . As SMN2 is present in all SMA
patients it has been extensively studied and serves as a drug target
for drugs that specifically correct splicing of exon 7 and thereby
increases amounts of functional SMN protein . As such,
large animal models where broader effects of both early and late
treatment can be carefully examined are becoming increasingly
relevant. In particular, the bioavailability and therapeutic potential
of drug candidates are more easily studied in animal models that
more closely resemble the physiology and metabolism of humans.
Transgenic models which have been generated through a
knockout/knock-in approach can potentially display pathologies
unrelated to the trans-gene itself, but as a consequence of gene
disruption caused by the insertion. This was recently reported in
the widely used Tg(SMN2)89Ahmb mouse model of SMA .
In order to construct a transgenic pig which resembles the
human SMA genotype as closely as possible we chose to study the
potential in converting the endogenous pig Smn1 to that of a
human SMN2 and in the process changing as little as possible in
the endogenous gene.
The aberrant splicing of human SMN2 exon 7 is caused by the
loss of an exonic splicing enhancer (ESE) due to a +6C.T
transition in SMN2 exon 7 relative to SMN1 exon 7, leading to loss
of binding of SRSF1 and increased binding of hnRNP A1 due to
strengthening of pre-existing exonic splicing silencer (ESS) motifs
[12,14,18,19]. In humans, the active ESE motif is altered from
CAGACAA to an inactive TAGACAA motif in SMN2, but in pigs
the ESE motif is only slightly altered to CAAACAA in the wild
type Smn1. This poses the question of whether or not this sequence
constitutes an active ESE and if a single Smn2-like +6C.T
mutation in porcine Smn1 exon 7 can disrupt the function and
result in a porcine Smn2-like gene.
We began by sequencing the Yucatan mini-pig Smn1 gene from
genomic DNA by designing primers to amplify individual exons
based on publicly available data as well as larger parts of the
intronic regions surrounding exon 7 which were not publicly
available at the time. Additionally, we performed 59RACE and
39RACE in order to validate previous assignment of exons and
UTR regions. The resulting Yucatan Smn1 gene sequence has
been submitted to the GenBank sequence database under
accession number KF585502. Contrary to previously published
data , but in line with the recently published Duroc porcine
genome from Ensembl (Sscrofa9), we found that porcine Smn1 is
composed of not 9 exons but instead 10, and that the last two
exons are located much further downstream from exon 7 than in
the human SMN1 and SMN2 genes.
In humans, exon 7 is followed by a 444 bp long intron but in
other animals, such as the mouse, intron 7 is much longer and the
final exon, exon 8, is completely different from the human
sequence. In pigs, however, exon 8 is a small 26 bp long exon
located 8.9 kb downstream from exon 7 and is then followed by a
3 kb long intron 8 after which the porcine Smn1 exon 9 begins
(Fig. 1A). Despite the significant difference in intron lengths,
porcine exon 9 and murine exon 8 seem to be homologous. One
striking observation though, is the overall similarity between
human SMN1/SMN2 and porcine Smn1 in terms of exon-intron
structure, with the exception of the 39 end of the transcripts, while
the intron lengths are very different in murine Smn1.
It is interesting to note that the natural stop-codon of porcine
Smn1 is located in the third to last exon, but successfully escapes
the NMD pathway (reviewed in ) as the stop-codon is within
50 bp from the last exon-exon junction. It is tempting to speculate
on the mechanism by which the small exon 8 arose in pigs and
whether or not it was ever used as an exon in other species such as
In addition to the endogenous Smn1 gene, we also identified
Smn1 sequences derived from at least two separately processed
pseudogenes, likely stemming from insertion of reverse transcribed
mature mRNA (Fig. S1). Subsequent BLAST searches 
revealed the location of these two pseudogenes on chromosome
2 and 13. We did not find evidence for expression of transcripts
from these pseudogenes, and due to accumulated mutations it is
doubtful that they would lead to production of functional protein if
they were transcribed.
We next examined if the splicing of SMN2 exon 7 could be
reestablished in a pig genomic context by introducing the exon 7+
6 C.T mutation. First, we introduced the wild type pig sequence
and a pig sequence with a mutation corresponding to the human +
6C.T SMN2 mutation into the pSXN13 splicing reporter as
previously described [19,23,24] and transfected Yucatan pig
fibroblasts with these constructs and constructs harboring the
corresponding human sequences (Fig. 2A). This showed that the
pig ESE sequence has splicing enhancer function, which is slightly
weaker than the corresponding human ESE sequence, and that
these ESEs are functional in pig cells. We then introduced a
change corresponding to the human +6C.T SMN2 mutation in
the pig ESE sequence and observed that it decreased inclusion of
the test exon, similar to the human SMN2 sequence (Fig. 2B). Next
we disabled the previously described flanking ESS in the inserted
sequences by introducing an A.C mutation . These results
show that the ESE in the wild type pig sequence, although
functional, is slightly weaker than the ESE in the corresponding
human SMN1 ESE sequence. They also show that this ESE
activity in the pig sequence is decreased in the mutated pig
SMN2like sequence. Overall, the splicing patterns of pSXN13 constructs
containing the pig sequences are very similar to corresponding
constructs containing the human sequences (Fig. 2B), but it
appears that the pig ESE is slightly weaker than the human SMN1
ESE, and that the negative effect of the +6C.T SMN2 mutation is
weaker when introduced into the pig sequence.
To investigate the splicing of pig SMN exon 7, we introduced
the pig ESE motif into our SMN minigene  and replaced 25 bp
of downstream intron 7 sequence with the corresponding pig
sequence (Fig. 3A). The immediate upstream intron sequence
containing the poly-pyrimidine tract (PPT) and 39splice site (39ss) is
completely identical between pig and human. When we
introduced the +6C.T mutation into the pig sequence the
increase in exon 7 skipping was modest (Fig. 3B) and we
speculated that this could be explained by the fact, that the intron
splicing silencer (ISS) sequence in pig intron 7 is different from the
human ISS . In humans, the ISS contains two potential
hnRNP A1 binding motifs, a proximal cagcat sequence and a
distal aagtga sequence, but one of these, the proximal, is abrogated
in the pig (Fig. 1B, cagcat.catcat). The distal hnRNP A1 binding
site seems to be strengthened in the pig sequence (aagtga.cagtga).
Therefore we tested both the human ISS sequence and a mutated
human ISS sequence where both potential hnRNP A1 sites are
disrupted by 2A.C mutations, which have previously been shown
to disrupt hnRNP A1 binding  (Fig 3A, constructs 36).
Interestingly, insertion of the human ISS resulted in a modest
increase in pig exon 7 exclusion (Fig. 3B), indicating that the
human ISS sequence has a stronger negative effect on SMN exon 7
inclusion than the corresponding pig ISS sequence. Insertion of
the the mutated human ISS resulted in a very modest decrease in
pig SMN exon 7 exclusion indicating that the pig ISS has only a
very modest ISS activity (Fig. 3B).
We investigated the pig ISS sequence further in our human
SMN minigene containing the human SMN1 and SMN2 ESE
sequences. Also in this context the pig ISS does not inhibit
inclusion of SMN2 exon 7 as strongly as the human ISS, and the
splicing pattern of the constructs with the mutated human ISS are
indistinguishable from those with the pig ISS, indicating that the
pig ISS is nearly inactive (Fig. 3B).
To establish the interactions of the pig ESE and ISS with
proteins known to bind the corresponding motifs in humans, we
performed RNA-affinity purification experiments using HeLa
nuclear extracts and RNA oligonucleotides with the sequences that
Figure 1. Genomic structure of SMN1 genes in humans, pigs and mice. A) SMN1 pre-mRNA transcripts expressed in humans, pigs and mice.
Exons included in transcripts are numbered according to historical nomenclature. Presence of pseudoexons in processed introns are indicated in
dashed outline and coloured according to the species expressing transcripts including these exons. Introns are drawn to scale and indicated as lines,
exons are not drawn to scale and are indicated as boxes. Start-codon is indicated by ATG and stop-codon by TAA. B) The start-sequence of intron 7 in
humans, pigs and mice. Bases that differ from the human sequence are indicated in bold underline. The location of the human ISS is indicated in
harbor the wild type pig and human ESE and ISS motifs as well as
mutated versions (Fig. 4A).
When we examined the binding of hnRNP A1 and SRSF1 to
the ESE motifs, we observed binding of SRSF1 to the pig
(Smn1like) ESE, and much less binding of hnRNP A1 (Fig. 4B). The
binding of hnRNP A1 to the mutated (Smn2-like) pig ESE was only
slightly increased, indicating that reduced hnRNP A1 binding does
not explain why the pig ESE retains some functionality when the +
6C.T mutation is introduced. Importantly, the +6C.T mutated
pig ESE bound SRSF1 very poorly, indicating that the loss of ESE
activity observed in the PSXN13 construct could be due to loss of
SRSF1 affinity (Fig. 4B). Similarly, we observed a decrease in
binding affinity towards SRSF1 in the SMN2 (+6C.T) construct
relative to SMN1. These results therefore indicate that the
SRSF1binding ESE in human SMN1 exon 7 is retained in pig and several
other species with an identical motif (Fig. S2).
In silico analysis of the wild type pig ESE sequence versus the
mutant using the Human Splicing Finder  (Table 1 and Fig.
S3) revealed that an ESE element in the wild type pig Smn1-like
sequence was removed according to ESEfinder which estimated a
drop in SRSF1 and SRSF2 score to below threshold in the
Smn2like ESE, indicating that SRSF1 and SRSF2 are proteins likely to
bind the wild type pig Smn1 ESE motif but not to the mutant
Smn2like motif [27,28]. The RESCUE-ESE algorithm similarly
indicated loss of an ESE motif  and the PESE algorithm also
reported a drop in score to below threshold [30,31]. Additionally,
new ESS motifs appeared in the mutant sequence according to the
FAS-ESS algorithm  and the IIE algorithm .
Using an intronic sequence spanning the full ISS motif as bait
(Fig. 4C) we observed strong binding of hnRNP A1 to the human
wild type ISS and reduced binding when the human ISS is
mutated in both hnRNP A1 sites (Fig.4D), in agreement with
earlier findings . The wild type pig ISS on the other hand,
displayed affinity towards hnRNP A1 to only a slightly lower
degree than the human wild type ISS. A mutation abrogating the
distal hnRNP A1 site in the pig ISS resulted in greatly reduced
binding of hnRNP A1, indicating that hnRNP A1 binding to the
pig ISS is mediated by the distal site (Fig 4D).
Initially, we sequenced the porcine Smn1 gene from the Yucatan
minipig, focusing on exonic sequences and intronic sequences
surrounding the exons, and in particular exon 7. Others have
previously published the sequence of the porcine Smn1 gene 
and the Sus scrofa genome is now available, but these sequences did
not originate from the Yucatan subspecies. We therefore
sequenced the Yucatan Smn1 exons and parts of intron 6 and
intron 7 in which we planned to position homology arms in order
to edit the endogenous Smn1 exon 7. This was prompted by the
observation that even small differences between different
subspecies can affect the efficiency of homologous recombination .
We observed that in agreement with the Sus scrofa reference
genome, the pig Smn1 gene contains a small 26 bp exon 8 which
differentiates it from both the human SMN1 gene and the murine
Smn1 gene. It is likely that the exon was activated by accumulation
of mutations within intron 7 of the ancestral Smn1 in the pig
Figure 2. Splicing analysis of pSXN13 minigenes. A) Summary of the pSXN13 minigene and the mutations introduced in the different
constructs. The hnRNP A1 binding sites within ISS-N1 have been indicated in dashed outline. Capitals indicate exonic bases. Bold italic bases in blue
indicate introduced mutations. The +6C.T mutation is indicated in bold italic red. The +8G.A mutation in pigs is indicated in underlined bold.
Construct numbers correspond to lane numbers in B. B) Representative RT-PCR results following transfection of Yucatan fibroblasts with minigene
constructs. Inclusion expressed as a percentage is indicated in the barplot, error bars indicate standard error of mean, n = 3. Lane numbers
correspond to construct numbers in A.
genome since this exon has not been observed in other species, but
other scenarios may also explain why pig Smn1 has an extra exon
compared to humans. Since the exon is positioned downstream of
the reading frame, and short enough for the NMD pathway not to
be activated, it doesnt impact the expression of the SMN protein,
but it is possible that it could lead to differences in the inclusion of
Smn1 exon 7 in the pig compared to humans and mice. We have
not addressed this scenario in this study, but consider it unlikely
that this exon would affect inclusion of exon 7 in a significantly
negative way as pigs, like mice, only have one SMN producing
gene. It is more likely that the greatly increased length of intron 7
in pigs would increase inclusion of exon 7 as it would delay the
competition between exon 7 and exon 8, giving the spliceosome
more time to recognize and process exon 7. This hypothesis is
supported by previous studies showing that inhibition of exon 8
splicing enhances inclusion of SMN2 exon 7 .
Another possibility is that pig exon 8 contains regulatory motifs
such as miRNA binding sites. However, we consider this possibility
unlikely since human SMN1 and mouse Smn1 transcripts have very
dissimilar 39UTR regions indicating that miRNA regulation
through motifs located in the 39UTR is unlikely to be involved
in the regulation of SMN expression.
The ESE motif in pig Smn1 exon 7 is slightly altered when
compared to the human motif but is identical to the ESE motif
found in other species such as cat and dog (Fig. S2). Notably, this
ESE motif does not contain a G nucleotide and should, in theory,
not constitute an hnRNP A1 binding site even when a +6 C.T
mutation is introduced in exon 7. Our results support this
hypothesis as we observed very low binding of hnRNP A1 to the
pig ESE motif compared to the human ESE motif.
In pig Smn1 intron 7 we also observed several differences in the
sequence compared to humans, in particular, the previously
Figure 3. Splicing analysis of SMN minigenes. A) Summary of the SMN minigene and the mutations introduced in the different constructs. The
hnRNP A1 binding sites within ISS-N1 have been indicated in dashed outline. Capitals indicate exonic bases. Bold underlined bases are bases that
differ between humans and pigs. Bold italic bases in blue are mutations introduced. The +6C.T mutation is indicated in bold italic red. Dots within
the sequence indicate a gap spanning multiple bases. Construct numbers correspond to lane numbers in B. B) RT-PCR results following transfection
of Yucatan fibroblasts with minigene constructs. Inclusion expressed as a percentage is indicated in the barplot, error bars indicate standard error of
mean, n = 3. Lane numbers correspond to construct numbers in A.
identified ISS [25,38] contained several substitutions that might
lead to altered function when compared to the human ISS.
We first investigated the ESE potential of the CAAACAA
sequence found in pig Smn1 exon 7 to evaluate the feasibility of
constructing a pig Smn2-like model by introducing a single +6C.T
mutation in the endogenous porcine Smn1 gene.
We introduced the ESE region into the pSXN13 splicing
reporter minigene and observed minimal inclusion of the
alternative exon in the wild type construct and complete loss of
inclusion when we introduced a C.T mutation analogous to the +
6T in SMN2. This demonstrates splicing enhancer activity of the
altered pig ESE and further demonstrates that a C.T mutation
can abrogate this activity despite the lack of an AG dinucleotide
within the ESE which has previously been proposed to form an
hnRNP A1 ESS in SMN2 exon 7. Therefore, in this context the
pig ESE supports the ESE-loss model. When we disrupted the
upstream ESS motif by an A.C mutation, the splicing pattern of
the wild type pig ESE and mutant pig ESE was very similar to that
of SMN1 and SMN2. However, the inclusion level of the wild type
pig Smn1-like ESE construct seemed slightly lower than that of the
wild type SMN1 construct, while the inclusion level of the mutant
pig Smn2-like ESE construct seemed slightly higher than that of the
SMN2 construct. These results can be explained by the ESE-loss/
ESS-gain model. The G.A change in the pig ESE seems to have
decreased the ESE activity relative to the human SMN1 ESE
sequence, but in the context of the +6C.T mutation, which
completely abolishes the human SMN1 ESE activity, the activity of
the gained ESS has also been decreased. Overall, the regulatory
Figure 4. RNA affinity pull-down experiments. A) RNA oligos spanning the ESE in SMN1 exon 7. Bases in bold indicate positions where the pig
and the human sequences differ. The +6C.T mutation is indicated in bold underline. X indicates biotin. B) Western blots of protein pull-downs with
oligos spanning the ESE. Two bands are seen for hnRNP A1, the upper band most likely being the alternative B splice isoform. Barplots indicate
normalized band intensities for the indicated protein bands. Error bars indicate standard error of mean (n = 3). C) RNA oligos spanning the ISS in
SMN1 intron 7. Bases in bold indicate positions where the pig sequence differs from the human. Mutations introduced are indicated in bold underline.
X indicates biotin. The hnRNP A1 binding sites within ISS-N1 have been indicated in dashed outline. D) Western blots of protein pull-downs with
oligos spanning the ISS. Barplots indicate normalized band intensities for the indicated protein bands. Error bars indicate standard error of mean
(n = 3).
element is more neutral in the pig Smn1 gene than in human SMN1
and SMN2, although it retains some ESE activity.
When we introduced pig Smn1 exon 7 with flanking regions in
our SMN model minigene, we observed only a modest decrease in
exon inclusion between the construct with the wild type sequence
and the +6C.T mutant construct (Fig. 3B), indicating that in the
native pig Smn1 gene a functional ESE is not crucial for exon 7
We then examined pig intron 7 and found that in the region of
the previously reported IVS7-ISS , there were differences
between the human and pig sequence which could potentially alter
binding affinity of one or both of the hnRNP A1 sites contained
within the ISS motif. In fact, the motif score was decreased for the
proximal hnRNP A1 site and increased for the distal site through
an A.C mutation which has previously been shown to increase
skipping of human SMN2 exon 7 .
These findings then lead us to investigate the ISS activity of the
downstream region in pig Smn1 intron 7 and we found that when
we inserted the human ISS into the pig context, a splicing pattern
more similar to SMN2 splicing pattern was observed when we
introduced the +6C.T mutation into the pig ESE sequence
(Fig. 3B). When we introduced mutations previously reported to
remove ISS activity , we observed exon inclusion similar to the
wild type pig construct (Fig. 3B). It seems that the inhibitory
function of the ISS is mostly dependent on the proximal hnRNP
A1 site and despite strengthening of the distal hnRNP A1 site in
The in silico analysis results of the wild-type pig ESE (+6C, CAAACAA) and the Smn2-like mutation (+6T, TAAACAA).
the pig ISS, it is not enough to overcome the loss of the proximal
When we examined the function of SMN1 and SMN2 ESE
sequences, we observed that the shift in exon inclusion was more
pronounced between SMN1 and SMN2 than the equivalent +6C.
T mutation in the pig ESE. One possible explanation is that the
mutated pig ESE still retains some ESE activity whereas the SMN2
ESE is completely inactivated. Another explanation could be that
because the pig ESE does not have a central G it is not able to
function as a low-affinity hnRNP A1 site and therefore it does not
contribute negatively to the inclusion of exon 7 to the same extent
as the inactivated SMN2 ESE.
To determine whether the mutated pig ESE binds SRSF1 more
efficiently than the SMN2 ESE and if it binds hnRNP A1 less
efficiently, we performed RNA-affinity purification experiments
with RNA oligonucleotides spanning the ESE region. We observed
strong binding of SRSF1 to the ESE motifs and much less to the
mutated motifs in both pig and human context indicating that
SRSF1 may enhance splicing of both human SMN1 and porcine
Smn1, but also that the residual ESE activity of the mutated pig
ESE may be through the binding to another SR protein. There
was also diminished hnRNP A1 binding to both the wild type pig
ESE and the mutated pig ESE. The reason is likely that the
SRSF1-binding ESE region in pig Smn1 constitutes an AC rich
element. These elements have previously been shown to function
as ESEs  and they do not contain any AG di-nucleotides,
which we and others have demonstrated to be essential for efficient
hnRNP A1 binding [19,40,41].
In silico analysis identified SRSF1 and SRSF2 as possible
proteins binding to an ESE in the pig wild type sequence, but not
the mutant sequence. However, since the SRSF2 motif and the 39
splice site are juxtaposed, it is not likely to be acting as a splice
enhancer if it is indeed functional. Although we did not observe
binding of SRSF1 to the mutated pig ESE at a level comparable to
the SMN1 ESE, this construct is artificial and although it may
have residual ESE activity through binding to another SR protein,
it does not necessarily follow that this SR protein is binding to the
natural pig ESE in vivo. SRSF1 therefore remains a likely candidate
as the SR protein binding to the ESE motif in pigs as well.
While the in silico analysis indicated that the porcine ESE
activity was diminished when the ESE was mutated to an
Smn2like motif (Table 1), we did not observe a pronounced increase in
exon skipping in the mutant constructs. In the pSXN13 reporter
minigene constructs, however, the pig ESE behaved almost
identically to the human ESE indicating that some ESE activity
is indeed lost. These results indicate that in the context of pig Smn1
exon 7, a strong ESE at position +6 is not a requirement for
efficient splicing, but it may still contribute to a stronger definition
of the exon.
The RNA-protein affinity studies of the IVS7-ISS pig motif
revealed overall similar binding of hnRNP A1 to the motif,
compared to the human wild type IVS7-ISS, despite a putative
increase in the strength of the distal hnRNP A1 core motif. This is
most likely caused by the disruption of the proximal motif which
may result in a complete loss of hnRNP A1 binding to that site.
The loss of this site is likely enough to effectively disrupt the
function of the entire ISS in the context of pig Smn1 exon 7. This is
in line with previous evidence that suggests a more central role of
the proximal site  compared to the distal site. In the context of
human SMN2 exon 7, abrogation of this site improves the
inclusion of exon 7, indicating that this site is a major contributor
to the inefficient splicing of human SMN2 exon 7. Additionally, the
IVS7-ISS has proved a valuable therapeutic target and has
resulted in on-going clinical trials with an ASO specifically
blocking this ISS motif [16,17,42].
Together, these findings indicate that in pigs, the IVS7-ISS is
inactive due to a mutation in the proximal hnRNP A1 site and this
inactivation has relaxed the requirements for a strong ESE at the
39ss of exon 7. This indicates that insertion of an SMN2-like
mutation in the endogenous porcine Smn1 gene would be
insufficient to recapitulate the splicing of SMN2 exon 7 and that
insertion of a larger fragment of the human SMN2 gene would be
In the mouse, the SRSF1 ESE is identical to the human ESE
but like the pig, the mouse IVS7-ISS is abrogated in the proximal
site (Fig. 1B). Furthermore, the distal site does not appear to be
strengthened indicating that the ISS is likely to be functionally
weakened also in the mouse. These observations indicate that the
human ISS would have to be inserted into the murine Smn1 gene if
Smn1 exon 7 skipping was to be induced by an +6C.T mutation,
but a mouse model containing a murine Smn1 to Smn2-like
conversion has been published without insertion of the human ISS
. This model exhibited a milder SMA phenotype similar to
human SMA type III, indicating that even though the Smn2-like
exon 7 was skipped, the level of inclusion was still higher than
human SMN2 exon 7, resulting in a mild phenotype. One possible
explanation for this, is that the murine ISS contains a 3 bp deletion
which moves the distal site in closer proximity to the 59ss, although
not as close as the proximal site of the human ISS. This indicates
that the inhibitory function of this hnRNP A1 core motif may be
highly dependent on its proximity to the 59ss and that it may
function by directly blocking access of U1 snRNP to the 59ss, and
not by inducing a polymerization of hnRNP A1 proteins along
exon 7, or by looping out the exon through interaction with other
ISS/ESS elements, such as the hnRNP A1 binding motif spanning
the 39ss of exon 7 or the hnRNP A1 motif generated across the
previous SRSF1 binding ESE in SMN2 [18,19].
In conclusion, we established the presence of a functional and
likely SRSF1 binding ESE in porcine Smn1, that this ESE may be
disrupted by a mutation similar to the +6C.T transition found in
SMN2 exon 7, and that porcine Smn1 is not dependent on activity
of this ESE due to the absence of a functional ISS motif in the
region immediately down-stream of porcine Smn1 exon 7. These
findings illustrate how key regulatory motifs may be switched on
and off in a specific order and that ESE motifs may be more
evolutionary conserved than ISS motifs, particularly in the context
of indispensable exons.
Furthermore, we found that splicing of porcine Smn1 is similar
to SMN1 in many ways, but that the regulation also differs
significantly. Crucially, the highly relevant therapeutic target
region containing ISS-N1 is disrupted in pigs and even if skipping
of porcine Smn1 could be induced by a single mutation,
therapeutics targeting this ISS would not be efficacious in this
We therefore suggest that porcine Smn1 may be converted to a
Smn2-like gene through insertion of a region spanning the end of
intron 6, the full exon 7 and intron 7, and the beginning of exon 8
of human SMN2 via techniques such as homologous
recombination. This would produce a hybrid SMN protein with a slightly
different C-terminal sequence derived from the human exon 7, but
we do not believe that this would significantly impact protein
function, as the C-terminal has previously been demonstrated to
be less dependent on the exact amino-acid sequence .
Materials and Methods
The minigenes used in this study were based on the SMN
human minigene described previously  and the pSXN13
splicing reporter minigene also previously described [19,23,24].
SMN minigenes. All SMN minigenes were ordered from and
prepared by Genscript (Piscataway, NJ, USA).
pSXN13 minigenes. To generate pSXN13 constructs we used
sense and antisense oligonucleotides with desired sequences, which
were mixed 1:1, phosphorylated and ligated to the BamHI and
SalI sites in the artificial exon within pSXN13 . The sequences
of the generated plasmids were verified by DNA sequencing.
Transient transfection of Yucatan fibroblasts and splicing
App. 2.06105 Yucatan fibroblasts were seeded in 6 well plates
and the following day transfected with 0.8 mg expression plasmid,
either SMN constructs or pSXN13 constructs, using FuGENE6
transfection reagent (Roche, Mannheim, Germany). Transfections
were performed in biological triplicates using fibroblasts from
Yucatan minipigs. 48 hours post transfection cells were washed in
1 x PBS-EDTA, lysed by adding 900 mL TRIzol reagent
(Invitrogen Co., Carlsbad, CA) and incubated on ice for 10 min
prior to RNA extraction according to the manufacturers
instructions (Invitrogen). Purified total RNA was used as template
in first strand cDNA synthesis using the Advantage MMLV
RTPCR kit (BD Biosciences Clontech, Franklin Lakes, NJ) with an
oligo (dT)18 primer. App. 1/10 of the cDNA synthesis product
corresponding to 100 ng RNA was used as template in each PCR
reaction using Tempase DNA polymerase (Ampliqon Aps,
Skovlunde, Denmark). For the SMN constructs we used an
exonexon junction spanning primer
(CATTCCAGAGAACTGTGGAGGT) and a primer located in SMN1/2 exon 8
(GTGGTGTCATTTAGTGCTGCTC). For the pSXN13
constructs we used a primer located in b-actin exon 1
(AAGGTGAACGTGGATGAAGTTGGTGGTG) and an exon-exon
junction spanning primer
(CCCACGTGCAGCCTTTGACCTAGTA). PCR products were separated and visualized on
agarose gels containing ethidium bromide (EtBr) on an Epi II
Darkroom UVP Transilluminator. Bands were quantitated by
optical densitometry using ImageJ 1.47  and normalized to the
length of the PCR product. Subsequently inclusion percentage was
estimated as the normalized intensity of the upper band divided by
the sum of the normalized intensities of the upper and lower band.
Amplification and sequencing of Yucatan minipig
Genomic DNA from Yucatan fibroblasts was extracted and
40 ng used as template in PCR reactions using Pfu polymerase
(Promega, Madison, WI, USA) with primers listed in table S1.
Amplified PCR products were sequenced on the ABI Prism
3100-Avant (Applied Biosystems, Foster City, CA). The partially
complete Yacatan minipig Smn1 sequence was submitted to
GenBank database under accession number KF585502.
The 59RACE and 39RACE experiments were carried out
according to the manufacturers instructions using the SMARTer
RACE kit (BD Biosciences Clontech).
Protein pull down by biotin coupled RNA
We used 39 biotinylated RNA oligonucleotides spanning either
the ESE motif: SMN1: 59-GGUUUCAGACAAAAUCAA-biotin,
SMN2: 59-GGUUUUAGACAAAAUCAA-biotin, pigSmn1:
59-GGUUUUAAACAAAAUCAA-biotin or the ISS motif: ISShum wt:
59CCAGCATTATGAAAGTGAA-biotin, ISShum mut:
59-CCCGCATTATGAACGTGAA-biotin, ISSpig wt:
59-CCATCATTATAACAGTGAA-biotin, ISSpig mut:
59-CCATCATTATAACCGTGAA-biotin. Pull-down assays and immunodetection of
purified proteins were carried out as previously described  in
three separate experiments using three separate nuclear extracts.
For each purification, 100 pmol of RNA oligonucleotide was
coupled to 100 ml of streptavidin-coupled magnetic beads (Dynal)
for 15 min in 1xbinding buffer (20 mM Hepes/KOH [pH 7.9],
72 mM KCl, 1.5 mM MgCl, 1.56 mM MgAc, 0.5 mM DTT,
4 mM glycerol, 0.75 mM ATP, and 0.2 mg/ml bulk tRNA). The
suspension was then placed in the magnet, and the supernatant
removed. The oligonucleotide-bead complexes were then
resuspended in 500 ml 1xbinding buffer containing 100 mL nuclear
extract purified from HeLa cells over-expressing either hnRNP A1
or SRSF1 and incubated 25 min at room temperature. Then, the
supernatant was removed, and the beads were washed three times
in 500 ml 1xbinding buffer containing 300 mM KCl. Finally, the
proteins bound to the RNA were eluted by the addition of 50 ml
protein sample buffer and heating for 4 min at 90uC.
Subsequently, 12 ml of the protein eluates were run on a 415% SDS-gel
and western blotted using mon-clonal antibodies against hnRNP
A1 (R9778, Sigma-Aldrich, Saint Louis, MO, USA) and SRSF1
(324500, Invitrogen). Bands were quantitated by optical
densitometry using ImageJ 1.47  and normalized to the geometric
mean band intensity per protein.
In silico analysis
We used Human Splicing Finder  to examine the sequences
for ESEs [2731,33] and ESSs [3033,46]. Wild-type and mutant
sequence covering the last 3 bp of intron 6 and the first 15 bp of
exon 7 were submitted using mutation analysis as the selected
analysis mode. Sequence alignments were done using MAFFT
. SMN1 exon 7 orthologue sequences were extracted from the
Ensembl database including 20 bp upstream and 30 bp
downstream of exon 7.
Figure S2 Multiple alignment of SMN1 exon 7 and mammalian
orthologues. MAFFT multiple alignment of SMN1 exon 7 with
spanning intronic sequences and seven mammalian orthologues.
Capitals indicate exonic bases, dashes indicate gaps. Asterisks
indicate fully conserved bases while periods indicate partially
Figure S3 Analysis of human SMN1 and porcine Smn1.
Graphical output of analysis of the ESE region in human SMN1
and porcine Smn1 using Human Splicing Finder. A drop in ESE
strength is indicated, as well as gain of a putative ESS in the
context of porcine Smn1 relative to human SMN1.
Table S1 Primers used for amplification of the porcine Smn1
gene from genomic DNA from Yucatan fibroblast. The listed
primers were used for PCR amplification using genomic DNA
from Yucatan fibroblasts as template.
We are grateful to Prof. Niels Gregersen for use of the ABI sequencer, and
to Jane Serup Pedersen, Lone Sundahl and Tudlik Emma Marie Andersen
for expert technical assistance.
Conceived and designed the experiments: TKD CBS BSA. Performed the
experiments: TKD LDS HSA SB AK. Analyzed the data: TKD CBS BSA.
Contributed reagents/materials/analysis tools: BSA CBS. Wrote the
paper: TKD HSA SB CBS BSA.
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